Human monoclonal antibodies to protein tyrosine kinase 7 (ptk7) and methods for using anti-ptk7 antibodies

ABSTRACT

The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to PTK7 with high affinity. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for detecting PTK7, as well as methods for treating various diseases, including cancer and infectious diseases, using anti-PTK7 antibodies.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/095,986, filed on Nov. 14, 2008, which is a U.S. 371 national phaseof International Patent Application No.: PCT/US2006/046837, filed onDec. 8, 2006, which claims the benefit of U.S. Provisional ApplicationNo. 60/748,373, filed on Dec. 8, 2005, the entire contents of which areincorporated herein in their entirety by this reference.

BACKGROUND OF THE INVENTION

Receptor tyrosine kinases (RTKs) are transmembrane signaling proteinsthat transmit biological signals from the extracellular environment tothe interior of the cell. The regulation of RTK signals is important forregulation of cell growth, differentiation, axonal growth, epithelialgrowth, development, adhesion, migration, and apoptosis (Prenzel et al.(2001) Endocr. Relat. Cancer 8:11-31; Hubbard and Till (2000) Annu. Rev.Biochem. 69:373-98). RTKs are known to be involved in the developmentand progression of several forms of cancer. In most of the RTK-relatedcancers, there has been an amplification of the receptor protein ratherthan a mutation of the gene (Kobus and Fleming (2005) Biochemistry44:1464-70).

Protein tyrosine kinase 7 (PTK7), a member of the receptor proteintyrosine kinase family, was first isolated from normal human melanocytesand cloned by RT-PCR (Lee et al., (1993) Oncogene 8:3403-10; Park etal., (1996) J. Biochem 119:235-9). Separately, the gene was cloned fromhuman colon carcinoma-derived cell lines and named colon carcinomakinase 4 (CCK4) (Mossie et al. (1995) Oncogene 11:2179-84). PTK7 belongsto a subset of RTKs that lack detectable catalytic tyrosine kinaseactivity but retain signal transduction activity and is thought topossibly function as a cell adhesion molecule.

The mRNA for PTK7 was found to be variably expressed in colon carcinomaderived cell lines but not found to be expressed in human adult colontissues (Mossie et al., supra). PTK7 expression was also seen in somemelanoma cell lines and melanoma biopsies (Easty, et al. (1997) Int. J.Cancer 71:1061-5). An alternative splice form was found to be expressedin hepatomas and colon cancer cells (Jung et al. (2002) Biochim BiophysActa 1579: 153-63). In addition, PTK7 was found to be highlyoverexpressed in acute myeloid leukemia samples (Muller-Tidow et al.,(2004) Clin. Cancer Res. 10:1241-9). By immunohistochemistry, tumorspecific staining of PTK7 was observed in breast, colon, lung,pancreatic, kidney and bladder cancers, as described in PCT PublicationWO 04/17992.

Accordingly, agents that recognize PTK7, and methods of using suchagents, are desired.

SUMMARY OF THE INVENTION

The present invention provides isolated monoclonal antibodies, inparticular human monoclonal antibodies, that bind to PTK7 and thatexhibit numerous desirable properties. These properties include highaffinity binding to human PTK7 and binding to Wilms' tumor cells. Alsoprovided are methods for treating a variety of PTK7 mediated diseasesusing the antibodies and compositions of the invention.

In one aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, wherein the antibody:

-   -   (a) specifically binds to human PTK7; and    -   (b) binds to a Wilms' tumor cell line (ATCC Acc No. CRL-1441).

Preferably the antibody is a human antibody, although in alternativeembodiments the antibody can be a murine antibody, a chimeric antibodyor humanized antibody.

In more preferred embodiments, the antibody binds to Wilms' tumor cellswith an EC₅₀ of 4.0 nM or less or binds to Wilms' tumor cells with anEC₅₀ of 3.5 nM or less.

In another embodiment, the antibody binds to a cancer cell line selectedfrom the group consisting of A-431 (ATCC Acc No. CRL-1555), Saos-2 (ATCCAcc No. HTB-85), SKOV-3 (ATCC Acc No. HTB-77), PC3 (ATCC Acc No.CRL-1435), DMS 114 (ATCC Acc No. CRL-2066), ACHN (ATCC Acc No.CRL-1611), LNCaP (ATCC Acc No. CRL-1740), DU 145 (ATCC Acc No. HTB-81),LoVo (ATCC Acc No. CCL-229) and MIA PaCa-2 (ATCC Acc No. CRL-1420) celllines.

In another embodiment, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, wherein the antibodycross-competes for binding to PTK7 with a reference antibody, whereinthe reference antibody:

-   -   (a) specifically binds to human PTK7; and    -   (b) binds to a Wilms' tumor cell line (ATCC Acc No. CRL-1441).        In various embodiments, the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:5;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:6;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:7; or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:8;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:9; or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:10.

In one aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 3-30.3 gene, wherein the antibody specifically binds PTK7. Theinvention also provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) DP44 gene,wherein the antibody specifically binds PTK7. The invention alsoprovides an isolated monoclonal antibody, or an antigen-binding portionthereof, comprising a heavy chain variable region that is the product ofor derived from a human V_(H) 3-33 gene, wherein the antibodyspecifically binds PTK7. The invention further provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) L15 gene, wherein the antibody specifically binds PTK7. Theinvention further provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) A10 gene,wherein the antibody specifically binds PTK7. The invention furtherprovides an isolated monoclonal antibody, or an antigen-binding portionthereof, comprising a light chain variable region that is the product ofor derived from a human V_(K) A27 gene, wherein the antibodyspecifically binds PTK7. The invention further provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) L6 gene, wherein the antibody specifically binds PTK7.

A preferred combination comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:11;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:15;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:19;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:23;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:29;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:35.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:11;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:15;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:19;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:24;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:30;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:36.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:12;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:16;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:20;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:25;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:31;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:37.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:13;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:17;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:21;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:26;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:32;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:38.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:13;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:17;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:21;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:27;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:33;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:39.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:14;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:18;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:22;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:28;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:34;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:40.        Other preferred antibodies of the invention, or antigen binding        portions thereof comprise:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:5.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:6.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:7.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:8.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:9.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:10.

The antibodies of the invention can be, for example, full-lengthantibodies, for example of an IgG1 or IgG4 isotype. Alternatively, theantibodies can be antibody fragments, such as Fab or Fab′2 fragments, orsingle chain antibodies.

The invention also provides an immunoconjugate comprising an antibody ofthe invention, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. Theinvention also provides a bispecific molecule comprising an antibody, orantigen-binding portion thereof, of the invention, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of the invention and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by theinvention, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Moreover, theinvention provides a transgenic mouse comprising human immunoglobulinheavy and light chain transgenes, wherein the mouse expresses anantibody of the invention, as well as hybridomas prepared from such amouse, wherein the hybridoma produces the antibody of the invention.

In yet another aspect, the invention provides a method of treating orpreventing a disease characterized by growth of tumor cells expressingPTK7, comprising administering to the subject the antibody, orantigen-binding portion thereof, of the invention in an amount effectiveto treat or prevent the disease. The disease can be, for example,cancer, e.g., colon cancer (including small intestine cancer), lungcancer, breast cancer, pancreatic cancer, melanoma (e.g., metastaticmalignant melanoma), acute myeloid leukemia, kidney cancer, bladdercancer, ovarian cancer and prostate cancer.

In a preferred embodiment, the invention provides a method of treatingcancer in vivo using an anti-PTK7 antibody. The anti-PTK7 antibody maybe a murine, chimeric, humanized or human antibody. Examples of othercancers that may be treated using the methods of the invention includerenal cancer (e.g., renal cell carcinoma), glioblastoma, brain tumors,chronic or acute leukemias including acute lymphocytic leukemia (ALL),adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g.,Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNSlymphoma, T-cell lymphoma, Burkitt's lymphoma, anaplastic large-celllymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-celllymphomas, peripheral T-cell lymphomas, Lennert's lymphomas,immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL),entroblastic/centrocytic (cb/cc) follicular lymphomas cancers, diffuselarge cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy(AILD)-like T cell lymphoma and HIV associated body cavity basedlymphomas), embryonal carcinomas, undifferentiated carcinomas of therhino-pharynx (e.g., Schmincke's tumor), Castleman's disease, Kaposi'sSarcoma, multiple myeloma, Waldenstrom's macroglobulinemia and otherB-cell lymphomas, nasopharangeal carcinomas, bone cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, rectal cancer, cancer of the anal region, stomachcancer, testicular cancer, uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, cancer of the esophagus, cancerof the small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, solid tumors of childhood, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stemglioma, pituitary adenoma, epidermoid cancer, squamous cell cancer,environmentally induced cancers including those induced by asbestos,e.g., mesothelioma and combinations of said cancers.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all references, Genbank entries,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO:41) and amino acidsequence (SEQ ID NO:1) of the heavy chain variable region of the 3G8 and3G8a human monoclonal antibodies. The CDR1 (SEQ ID NO:11), CDR2 (SEQ IDNO:15) and CDR3 (SEQ ID NO:19) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO:45) and amino acidsequence (SEQ ID NO:5) of the light chain variable region of the 3G8human monoclonal antibody. The CDR1 (SEQ ID NO:23), CDR2 (SEQ ID NO:29)and CDR3 (SEQ ID NO:35) regions are delineated and the V and J germlinederivations are indicated.

FIG. 1C shows the nucleotide sequence (SEQ ID NO:46) and amino acidsequence (SEQ ID NO:6) of the light chain variable region of the 3G8ahuman monoclonal antibody. The CDR1 (SEQ ID NO:24), CDR2 (SEQ ID NO:30)and CDR3 (SEQ ID NO:36) regions are delineated and the V and J germlinederivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO:42) and amino acidsequence (SEQ ID NO:2) of the heavy chain variable region of the 4D5human monoclonal antibody. The CDR1 (SEQ ID NO:12), CDR2 (SEQ ID NO:16)and CDR3 (SEQ ID NO:20) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO:47) and amino acidsequence (SEQ ID NO:7) of the light chain variable region of the 4D5human monoclonal antibody. The CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:31)and CDR3 (SEQ ID NO:37) regions are delineated and the V and J germlinederivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO:43) and amino acidsequence (SEQ ID NO:3) of the heavy chain variable region of the 12C6human monoclonal antibodies. The CDR1 (SEQ ID NO:13), CDR2 (SEQ IDNO:17) and CDR3 (SEQ ID NO:21) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO:48) and amino acidsequence (SEQ ID NO:8) of the light chain variable region of the 12C6human monoclonal antibody. The CDR1 (SEQ ID NO:26), CDR2 (SEQ ID NO:32)and CDR3 (SEQ ID NO:38) regions are delineated and the V and J germlinederivations are indicated.

FIG. 3C shows the nucleotide sequence (SEQ ID NO:49) and amino acidsequence (SEQ ID NO:9) of the light chain variable region of the 12C6ahuman monoclonal antibody. The CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:33)and CDR3 (SEQ ID NO:39) regions are delineated and the V and J germlinederivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO:44) and amino acidsequence (SEQ ID NO:4) of the heavy chain variable region of the 7C8human monoclonal antibody. The CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:18)and CDR3 (SEQ ID NO:22) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO:50) and amino acidsequence (SEQ ID NO:10) of the light chain variable region of the 7C8human monoclonal antibody. The CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:34)and CDR3 (SEQ ID NO:40) regions are delineated and the V and J germlinederivations are indicated.

FIG. 5 shows the alignment of the amino acid sequences of the heavychain variable regions of 3G8 (SEQ ID NO: 1) and 3G8a (SEQ ID NO: 1)with the human germline V_(H) 3-30.3 amino acid sequence (SEQ ID NO:51)(JH4b germline disclosed as SEQ ID NO: 59).

FIG. 6 shows the alignment of the amino acid sequence of the heavy chainvariable region of 4D5 (SEQ ID NO: 2) with the human germline V_(H)3-30.3 amino acid sequence (SEQ ID NO:51) (JH4b germline disclosed asSEQ ID NO: 60).

FIG. 7 shows the alignment of the amino acid sequences of the heavychain variable regions of 12C6 (SEQ ID NO: 3) and 12C6a (SEQ ID NO: 2)with the human germline V_(H) DP44 amino acid sequence (SEQ ID NO:52)(3-7, 3-23, and JH4b germlines disclosed as SEQ ID NOS 61-63,respectively).

FIG. 8 shows the alignment of the amino acid sequence of the heavy chainvariable region of 7C8 (SEQ ID NO: 4) with the human germline V_(H) 3-33amino acid sequence (SEQ ID NO:53) (JH6b germline disclosed as SEQ IDNO: 64).

FIG. 9 shows the alignment of the amino acid sequences of the lightchain variable regions of 3G8 (SEQ ID NO: 5) and 3G8a (SEQ ID NO: 6)with the human germline V_(k) L15 amino acid sequence (SEQ ID NO:54)(JK1 germline disclosed as SEQ ID NO: 65).

FIG. 10 shows the alignment of the amino acid sequence of the lightchain variable region of 4D5 (SEQ ID NO: 7) with the human germlineV_(k) A10 amino acid sequence (SEQ ID NO:55) (JK5 germline disclosed asSEQ ID NO: 66).

FIG. 11 shows the alignment of the amino acid sequence of the lightchain variable region of 12C6 (SEQ ID NO: 8) with the human germlineV_(k) A27 amino acid sequence (SEQ ID NO:56) (JK2 germline disclosed asSEQ ID NO: 67).

FIG. 12 shows the alignment of the amino acid sequence of the lightchain variable region of 12C6a (SEQ ID NO: 9) with the human germlineV_(k) L15 amino acid sequence (SEQ ID NO:54) (JK2 germline disclosed asSEQ ID NO: 68).

FIG. 13 shows the alignment of the amino acid sequence of the lightchain variable region of 7C8 (SEQ ID NO: 10) with the human germlineV_(k) L6 amino acid sequence (SEQ ID NO:57) (JK3 germline disclosed asSEQ ID NO: 69).

FIG. 14 shows the results of flow cytometry experiments demonstratingthat the human monoclonal antibody 7C8, directed against human PTK7,binds the cell surface of HEK3 cells tranfected with full-length humanPTK7.

FIG. 15 shows the results of ELISA experiments demonstrating that humanmonoclonal antibodies against human PTK7 specifically bind to PTK7.

FIG. 16 shows the results of flow cytometry experiments demonstratingthat antibodies directed against human PTK7 binds the cell surface ofWilms' tumor cells.

FIG. 17 shows the results of flow cytometry experiments demonstratingthat antibodies directed against human PTK7 binds the cell surface of avariety of cancer cell lines.

FIG. 18 shows the results of flow cytometry experiments demonstratingthat antibodies directed against human PTK7 binds the cell surface ofdendritic cells.

FIG. 19 shows the results of flow cytometry experiments demonstratingthat antibodies directed against human PTK7 bind to CD4+ and CD8+T-lymphocytes, but not to B-lymphocytes.

FIG. 20 shows the results of Hum-Zap internalization experimentsdemonstrating that human monoclonal antibodies against human PTK7 caninternalize into PTK7+ cells. (A) Internalization of the humanantibodies 3G8, 4D5 and 7C8 into Wilms' tumor cells. (B) Internalizationof the human antibody 12C6 into Wilms' tumor cells. (C) Internalizationof the human antibodies 7C8 and 12C6 into A-431 tumor cells. (D)Internalization of the human antibodies 7C8 and 12C6 into PC3 tumorcells.

FIG. 21 shows the results of a cell proliferation assay demonstratingthat toxin-conjugated human monoclonal anti-PTK7 antibodies kill humankidney cancer cell lines.

FIG. 22 shows the results of a cell proliferation assay demonstratingthat toxin-conjugated human monoclonal anti-PTK7 antibodies kill celllines expressing low to high levels of PTK7 expression.

FIG. 23 shows the results of an invasion assay demonstrating thatanti-PTK7 antibodies inhibit the invasion mobility of cells expressingPTK7 on the cell surface.

FIG. 24 shows the results of an in vivo tumor xenograft studydemonstrating that anti-PTK7 antibodies conjugated to a toxin slowedtumor growth progression in pancreatic cancer.

FIG. 25 shows the results of an in vivo tumor xenograft studydemonstrating that anti-PTK7 antibodies conjugated to a toxin slowedtumor growth progression in breast cancer.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to isolated monoclonalantibodies, particularly human monoclonal antibodies, that bindspecifically to PTK7. In certain embodiments, the antibodies of theinvention exhibit one or more desirable functional properties, such ashigh affinity binding to PTK7 and/or the ability to inhibit growth oftumor cells in vitro or in vivo. In certain embodiments, the antibodiesof the invention are derived from particular heavy and light chaingermline sequences and/or comprise particular structural features suchas CDR regions comprising particular amino acid sequences. The inventionprovides isolated antibodies, methods of making such antibodies,immunoconjugates and bispecific molecules comprising such antibodies andpharmaceutical compositions containing the antibodies, immunconjugatesor bispecific molecules of the invention. The invention also relates tomethods of using the antibodies, such as to treat diseases such ascancer.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “PTK7” and “CCK4” are used interchangeably and includevariants, isoforms and species homologs of human PTK7. Accordingly,human antibodies of the invention may, in certain cases, cross-reactwith PTK7 from species other than human. In certain embodiments, theantibodies may be completely specific for one or more human PTK7 and maynot exhibit species or other types of non-human cross-reactivity. Thecomplete amino acid sequence of an exemplary human PTK7 has Genbankaccession number NM_(—)002821 (SEQ ID NO:58).

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present invention is the PTK7 receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., PTK7). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region(see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds PTK7 is substantially free of antibodies that specifically bindantigens other than PTK7). An isolated antibody that specifically bindsPTK7 may, however, have cross-reactivity to other antigens, such as PTK7molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds to human PTK7” isintended to refer to an antibody that binds to human PTK7 with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, more preferably1×10⁻⁸ M or less, more preferably 5×10⁻⁹ M or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M orless, even more preferably 10⁻⁹ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-PTK7 Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to PTK7. Preferably, an antibody of theinvention binds to PTK7 with high affinity, for example with a K_(D) of1×10⁻⁷ M or less. The anti-PTK7 antibodies of the invention preferablyexhibit one or more of the following characteristics:

-   -   (a) specifically binds to human PTK7; or    -   (b) binds to a Wilms' tumor cell line (ATCC Acc No. CRL-1441).        Preferrably, the antibody binds to human PTK7 with a K_(D) of        5×10⁻⁸ M or less, binds to human PTK7 with a K_(D) of 1×10⁻⁸ M        or less, binds to human PTK7 with a K_(D) of 5×10⁻⁹ M or less,        or binds to human PTK7 with a K_(D) of between 1×10⁻⁸M and        1×10⁻¹⁰ M or less. Preferrably, the antibody binds to Wilms'        tumor cells with an EC₅₀ of 4.0 nM or less, or binds to Wilms'        tumor cells with an EC₅₀ of 3.5 nM or less. Standard assays to        evaluate the binding ability of the antibodies toward PTK7 are        known in the art, including for example, ELISAs, Western blots        and RIAs. The binding kinetics (e.g., binding affinity) of the        antibodies also can be assessed by standard assays known in the        art, such as by ELISA, Scatchard and Biacore analysis. As        another example, the antibodies of the present invention may        bind to a kidney carcinoma tumor cell line, for example, the        Wilms' tumor cell line. Suitable assays for evaluating any of        the above-described characteristics are described in detail in        the Examples.

Monoclonal Antibodies 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8

Preferred antibodies of the invention are the human monoclonalantibodies 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8, isolated andstructurally characterized as described in Examples 1 and 2. Thosehaving ordinary skill in the art shall appreciate that the antibodies3G8 and 3G8a, as well as the antibodies 12C6 and 12C6a have the sameheavy chain sequence, while differing in their light chain sequences.The V_(H) amino acid sequences of 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8are shown in SEQ ID NOs: 1 (3G8 and 3G8a), 2 (4D5), 3 (12C6 and 12C6a)and 4 (7C8). The V_(L) amino acid sequences of 3G8, 3G8a, 4D5, 12C6,12C6a, and 7C8 are shown in SEQ ID NOs: 5, 6, 7, 8, 9 and 10,respectively.

Given that each of these antibodies can bind to PTK7, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-PTK7binding molecules of the invention. PTK7 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). Preferably, when V_(H) andV_(L) chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9 and 10;

wherein the antibody specifically binds PTK7, preferably human PTK7.Preferred heavy and light chain combinations include:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:1; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:5; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:1; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:6; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:2; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:7; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:3; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:8; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:3; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:9; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:4; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO:10.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 3G8, 3G8a, 4D5,12C6, 12C6a and 7C8, or combinations thereof. The amino acid sequencesof the V_(H) CDR1s of 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 are shown inSEQ ID NOs: 11 (3G8 and 3G8a), 12 (4D5), 13 (12C6 and 12C6a) and 14(7C8). The amino acid sequences of the V_(H) CDR2s of 3G8, 3G8a, 4D5,12C6, 12C6a and 7C8 are shown in SEQ ID NOs: (3G8 and 3G8a), 16 (4D5),17 (12C6 and 12C6a) and 18 (7C8). The amino acid sequences of the V_(H)CDR3s of 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 are shown in SEQ ID NOs: 19(3G8 and 3G8a), 20 (4D5), 21 (12C6 and 12C6a) and 22 (7C8). The aminoacid sequences of the V_(k) CDR1s of 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8are shown in SEQ ID NOs: 23, 24, 25, 26, 27 and 28, respectively. Theamino acid sequences of the V_(k) CDR2s of 3G8, 3G8a, 4D5, 12C6, 12C6aand 7C8 are shown in SEQ ID NOs: 29, 30, 31, 32, 33 and 34,respectively. The amino acid sequences of the V_(k) CDR3s of 3G8, 3G8a,4D5, 12C6, 12C6a and 7C8 are shown in SEQ ID NOs: 35, 36, 37, 38, 39 and40, respectively. The CDR regions are delineated using the Kabat system(Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242).

Given that each of these antibodies can bind to PTK7 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(k) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(k) CDR1, CDR2, and CDR3)to create other anti-PTK7 binding molecules of the invention. PTK7binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(k) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(k) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 3G8, 3G8a, 4D5,12C6, 12C6a and 7C8.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 12, 13 and 14;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 15, 16, 17 and 18;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19, 20, 21 and 22;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27 and28;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33 and34; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39 and40;

wherein the antibody specifically binds PTK7, preferably human PTK7.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:11;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:15;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:19;

(d) a light chain variable region CDR1 comprising SEQ ID NO:23;

(e) a light chain variable region CDR2 comprising SEQ ID NO:29; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:35.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:11;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:15;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:19;

(d) a light chain variable region CDR1 comprising SEQ ID NO:24;

(e) a light chain variable region CDR2 comprising SEQ ID NO:30; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:36.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:12;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:16;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:20;

(d) a light chain variable region CDR1 comprising SEQ ID NO:25;

(e) a light chain variable region CDR2 comprising SEQ ID NO:31; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:37.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:13;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:17;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:21;

(d) a light chain variable region CDR1 comprising SEQ ID NO:26;

(e) a light chain variable region CDR2 comprising SEQ ID NO:32; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:38.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:13;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:17;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:21;

(d) a light chain variable region CDR1 comprising SEQ ID NO:27;

(e) a light chain variable region CDR2 comprising SEQ ID NO:33; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:39.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:14;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:18;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:22;

(d) a light chain variable region CDR1 comprising SEQ ID NO:28;

(e) a light chain variable region CDR2 comprising SEQ ID NO:34; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:40.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka et al., British J. ofCancer 83(2):252-260 (2000) (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.296:833-849 (2000) (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader et al., Proc. Natl.Acad. Sci. U.S.A. 95:8910-8915 (1998) (describing a panel of humanizedanti-integrin α_(v)β₃ antibodies using a heavy and light chain variableCDR3 domain of a murine anti-integrin α_(v)β₃ antibody LM609 whereineach member antibody comprises a distinct sequence outside the CDR3domain and capable of binding the same epitope as the parent muringantibody with affinities as high or higher than the parent murineantibody); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994)(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci.U.S.A. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3sequences of three Fabs (SI-1, SI-40, and SI-32) against human placentalDNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacingthe existing heavy chain CDR3 and demonstrating that the CDR3 domainalone conferred binding specificity); and Ditzel et al., J. Immunol.157:739-749 (1996) (describing grafting studies wherein transfer of onlythe heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chainof a monospecific IgG tetanus toxoid-binding Fab p313 antibody wassufficient to retain binding specificity of the parent Fab). Each ofthese references is hereby incorporated by reference in its entirety.

Accordingly, the present invention provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domains from anantibody derived from a human or non-human animal, wherein themonoclonal antibody is capable of specifically binding to PTK7. Withincertain aspects, the present invention provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domain from anon-human antibody, such as a mouse or rat antibody, wherein themonoclonal antibody is capable of specifically binding to PTK7. Withinsome embodiments, such inventive antibodies comprising one or more heavyand/or light chain CDR3 domain from a non-human antibody (a) are capableof competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present invention provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a human antibody, such as, for example, a human antibody obtainedfrom a non-human animal, wherein the human antibody is capable ofspecifically binding to PTK7. Within other aspects, the presentinvention provides monoclonal antibodies comprising one or more heavyand/or light chain CDR3 domain from a first human antibody, such as, forexample, a human antibody obtained from a non-human animal, wherein thefirst human antibody is capable of specifically binding to PTK7 andwherein the CDR3 domain from the first human antibody replaces a CDR3domain in a human antibody that is lacking binding specificity for PTK7to generate a second human antibody that is capable of specificallybinding to PTK7. Within some embodiments, such inventive antibodiescomprising one or more heavy and/or light chain CDR3 domain from thefirst human antibody (a) are capable of competing for binding with; (b)retain the functional characteristics; (c) bind to the same epitope;and/or (d) have a similar binding affinity as the corresponding parentalfirst human antibody. In preferred embodiments, the first human antibodyis 3G8, #g8a, 4D5, 12C6, 12C6a or 7C8.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-30.3 gene, wherein the antibodyspecifically binds PTK7, preferably human PTK7. In another preferredembodiment, the invention provides an isolated monoclonal antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) DP44 gene,wherein the antibody specifically binds PTK7, preferably human PTK7. Inanother preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising aheavy chain variable region that is the product of or derived from ahuman V_(H) 3-33 gene, wherein the antibody specifically binds PTK7,preferably human PTK7. In yet another preferred embodiment, theinvention provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) L15 gene,wherein the antibody specifically binds PTK7, preferably human PTK7. Inyet another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) A10 gene, wherein the antibody specifically binds PTK7,preferably human PTK7. In yet another preferred embodiment, theinvention provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) A27 gene,wherein the antibody specifically binds PTK7, preferably human PTK7. Inyet another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) L6 gene, wherein the antibody specifically binds PTK7,preferably human PTK7. In yet another preferred embodiment, theinvention provides an isolated monoclonal antibody, or antigen-bindingportion thereof, wherein the antibody:

(a) comprises a heavy chain variable region that is the product of orderived from a human V_(H) 3-30.3, DP44 or 3-33 gene (which gene encodesthe amino acid sequence set forth in SEQ ID NOs: 51, 52 or 53,respectively);

(b) comprises a light chain variable region that is the product of orderived from a human V_(K) L15, A10, A27 or L6 gene (which gene encodesthe amino acid sequence set forth in SEQ ID NO:54, 55, 56 or 57,respectively); and

(c) specifically binds to PTK7.

Examples of antibodies having V_(H) and V_(K) of V_(H) 3-30.3 and V_(K)L15, respectively, are 3G8 and 3G8a. An example of an antibody havingV_(H) and V_(K) of V_(H) 3-30.3 and V_(K) A10, respectively is 4D5. Anexample of an antibody having V_(H) and V_(K) of V_(H) DP44 and V_(K)A27, respectively is 12C6. An example of an antibody having V_(H) andV_(K) of V_(H) DP44 and V_(K) L15, respectively is 12C6a. An example ofan antibody having V_(H) and V_(K) of V_(H) 3-33 and V_(K) L6,respectively is 7C8.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-PTK7 antibodies of the invention.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 1, 2,        3 and 4;    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 5, 6,        7, 8, 9 and 10; and    -   the antibody exhibits one or more of the following properties:    -   (c) the antibody binds to human PTK7 with a K_(D) of 1×10⁻⁷ M or        less;    -   (d) the antibody binds to the Wilms' tumor cell line.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, followedby testing of the encoded altered antibody for retained function (i.e.,the functions set forth in (c) and (d) above) using the functionalassays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g., 3G8,3G8a, 4D5, 12C6, 12C6a or 7C8), or conservative modifications thereof,and wherein the antibodies retain the desired functional properties ofthe anti-PTK7 antibodies of the invention. Accordingly, the inventionprovides an isolated monoclonal antibody, or antigen binding portionthereof, comprising a heavy chain variable region comprising CDR1, CDR2,and CDR3 sequences and a light chain variable region comprising CDR1,CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 19, 20, 21 and 22, and conservative modificationsthereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 35, 36, 37, 38, 39 and 40, and conservative modificationsthereof; and

-   -   the antibody exhibits one or more of the following properties:

(c) specifically binds to human PTK7; and

(d) binds to a Wilms' tumor cell line (ATCC Acc No. CRL-1441).

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 15, 16, 17 and 18, and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, andconservative modifications thereof. In another preferred embodiment, theheavy chain variable region CDR1 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs: 11, 12, 13 and 14, and conservative modifications thereof;and the light chain variable region CDR1 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 23, 24, 25, 26, 27 and 28, and conservative modificationsthereof.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c) and (d) above) using thefunctional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-PTK7 Antibodies of theInvention

In another embodiment, the invention provides antibodies that bind tothe same epitope on human PTK7 as any of the PTK7 monoclonal antibodiesof the invention (i.e., antibodies that have the ability tocross-compete for binding to PTK7 with any of the monoclonal antibodiesof the invention). In preferred embodiments, the reference antibody forcross-competition studies can be the monoclonal antibody 3G8 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 5,respectively), or the monoclonal antibody 3G8a (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 1 and 6, respectively), or themonoclonal antibody 4D5 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs: 2 and 7, respectively), or the monoclonal antibody 12C6(having V_(H) and V_(L) sequences as shown in SEQ ID NOs: 3 and 8,respectively), or the monoclonal antibody 12C6a (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 3 and 9, respectively), or themonoclonal antibody 7C8 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs: 4 and 10, respectively). Such cross-competing antibodies canbe identified based on their ability to cross-compete with 3G8, 3G8a,4D5, 12C6, 12C6a or 7C8 in standard PTK7 binding assays. For example,BIAcore analysis, ELISA assays or flow cytometry may be used todemonstrate cross-competition with the antibodies of the currentinvention. The ability of a test antibody to inhibit the binding of, forexample, 3G8, 3G8a, 4D5, 12C6, 12C6a or 7C8, to human PTK7 demonstratesthat the test antibody can compete with 3G8, 3G8a, 4D5, 12C6, 12C6a or7C8 for binding to human PTK7 and thus binds to the same epitope onhuman PTK7 as 3G8, 3G8a, 4D5, 12C6, 12C6a or 7C8. In a preferredembodiment, the antibody that binds to the same epitope on human PTK7 as3G8, 3G8a, 4D5, 12C6, 12C6a or 7C8 is a human monoclonal antibody. Suchhuman monoclonal antibodies can be prepared and isolated as described inthe Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 11, 12, 13 and 14, SEQ ID NOs: 15, 16, 17 and 18 and SEQ IDNOs: 19, 20, 21 and 22, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 sequences comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 23, 24, 25,26, 27 and 28, SEQ ID NOs: 29, 30, 31, 32, 33 and 34 and SEQ ID NOs: 35,36, 37, 38, 39 and 40, respectively. Thus, such antibodies contain theV_(H) and V_(L) CDR sequences of monoclonal antibodies 3G8, 3G8a, 4D5,12C6, 12C6a or 7C8 yet may contain different framework sequences fromthese antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference. As another example, the germline DNAsequences for human heavy and light chain variable region genes can befound in the Genbank database. For example, the following heavy chaingermline sequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank accession numbers: 1-69 (NG_(—)0010109,NT_(—)024637 and BC070333), 3-33 (NG_(—)0010109 and NT_(—)024637) and3-7 (NG_(—)0010109 and NT_(—)024637). As another example, the followingheavy chain germline sequences found in the HCo12 HuMAb mouse areavailable in the accompanying Genbank accession numbers: 1-69(NG_(—)0010109, NT_(—)024637 and BC070333), 5-51 (NG_(—)0010109 andNT_(—)024637), 4-34 (NG_(—)0010109 and NT_(—)024637), 3-30.3 (?) and3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research25:3389-3402), which is well known to those skilled in the art. BLAST isa heuristic algorithm in that a statistically significant alignmentbetween the antibody sequence and the database sequence is likely tocontain high-scoring segment pairs (HSP) of aligned words. Segment pairswhose scores cannot be improved by extension or trimming is called ahit. Briefly, the nucleotide sequences of VBASE origin(http://vbase.mrc-cpe.cam.ac.uldvbase1/list2.php) are translated and theregion between and including FR1 through FR3 framework region isretained. The database sequences have an average length of 98 residues.Duplicate sequences which are exact matches over the entire length ofthe protein are removed. A BLAST search for proteins using the programblastp with default, standard parameters except the low complexityfilter, which is turned off, and the substitution matrix of BLOSUM62,filters for top 5 hits yielding sequence matches. The nucleotidesequences are translated in all six frames and the frame with no stopcodons in the matching segment of the database sequence is consideredthe potential hit. This is in turn confirmed using the BLAST programtblastx, which translates the antibody sequence in all six frames andcompares those translations to the VBASE nucleotide sequencesdynamically translated in all six frames.

The identities are exact amino acid matches between the antibodysequence and the protein database over the entire length of thesequence. The positives (identities+substitution match) are notidentical but amino acid substitutions guided by the BLOSUM62substitution matrix. If the antibody sequence matches two of thedatabase sequences with same identity, the hit with most positives wouldbe decided to be the matching sequence hit.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H)3-30.3 framework sequences (SEQ ID NO:51) and/or the V_(H) DP44framework sequences (SEQ ID NO:52) and/or the V_(H) 3-33 frameworksequences (SEQ ID NO:53) and/or the V_(K) L15 framework sequences (SEQID NO:54) and/or the V_(K) A10 framework sequences (SEQ ID NO:55) and/orthe V_(K) L15 framework sequences (SEQ ID NO:54) and/or the V_(K) A27framework sequences (SEQ ID NO:56) and/or the V_(K) L15 frameworksequences (SEQ ID NO:54) and/or the V_(K) L6 framework sequences (SEQ IDNO:57) used by preferred monoclonal antibodies of the invention. TheV_(H) CDR1, CDR2, and CDR3 sequences, and the V_(K) CDR1, CDR2, and CDR3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-PTK7 monoclonal antibodies, or antigen binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 11, 12, 13 and 14, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 11, 12, 13 and 14; (b) a V_(H)CDR2 region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 15, 16, 17 and 18, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 15, 16, 17 and 18; (c) a V_(H)CDR3 region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19, 20, 21 and 22, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 19, 20, 21 and 22; (d) a V_(K)CDR1 region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23, 24, 25, 26, 27 and 28, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 23, 24, 25, 26, 27 and28; (e) a V_(K) CDR2 region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 29, 30,31, 32, 33 and 34; and (f) a V_(K) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 35, 36, 37,38, 39 and 40, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 35, 36, 37, 38, 39 and 40.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(K), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, for 3G8 (and 3G8a), amino acid residue #28 (within FR1) ofV_(H) is an isoleucine whereas this residue in the corresponding V_(H)3-30.3 germline sequence is a threonine. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis (e.g., residue #28 of FR1 of theV_(H) of 3G8 (and 3G8a) can be “backmutated” from isoleucine tothreonine).

As another example, for 12C6 (and 12C6a), amino acid residue #44 (withinFR2) of V_(H) is a threonine whereas this residue in the correspondingV_(H) DP44 germline sequence is a glycine. To return the frameworkregion sequences to their germline configuration, for example, residue#44 (residue #9 of FR2) of the V_(H) of 12C6 (and 12C6a) can be“backmutated” from threonine to glycine. Such “backmutated” antibodiesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1, FcγR11, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 by Hanai et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanai et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describescell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III(GnTIII)) such that antibodies expressed in the engineered cell linesexhibit increased bisecting GlcNac structures which results in increasedADCC activity of the antibodies (see also Umana et al. (1999) Nat.Biotech. 17:176-180). Alternatively, the fucose residues of the antibodymay be cleaved off using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Antibody Physical Properties

The antibodies of the present invention may be further characterized bythe various physical properties of the anti-PTK7 antibodies. Variousassays may be used to detect and/or differentiate different classes ofantibodies based on these physical properties.

In some embodiments, antibodies of the present invention may contain oneor more glycosylation sites in either the light or heavy chain variableregion. The presence of one or more glycosylation sites in the variableregion may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala F A andMorrison S L (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro R G (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. Variable region glycosylation may be tested using a Glycoblotassay, which cleaves the antibody to produce a Fab, and then tests forglycosylation using an assay that measures periodate oxidation andSchiff base formation. Alternatively, variable region glycosylation maybe tested using Dionex light chromatography (Dionex-LC), which cleavessaccharides from a Fab into monosaccharides and analyzes the individualsaccharide content. In some instances, it is preferred to have ananti-PTK7 antibody that does not contain variable region glycosylation.This can be achieved either by selecting antibodies that do not containthe glycosylation motif in the variable region or by mutating residueswithin the glycosylation motif using standard techniques well known inthe art.

In a preferred embodiment, the antibodies of the present invention donot contain asparagine isomerism sites. A deamidation or isoasparticacid effect may occur on N-G or D-G sequences, respectively. Thedeamidation or isoaspartic acid effect results in the creation ofisoaspartic acid which decreases the stability of an antibody bycreating a kinked structure off a side chain carboxy terminus ratherthan the main chain. The creation of isoaspartic acid can be measuredusing an iso-quant assay, which uses a reverse-phase HPLC to test forisoaspartic acid.

Each antibody will have a unique isoelectric point (pI), but generallyantibodies will fall in the pH range of between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.Antibodies may have a pI that is outside this range. Although theeffects are generally unknown, there is speculation that antibodies witha pI outside the normal range may have some unfolding and instabilityunder in vivo conditions. The isoelectric point may be tested using acapillary isoelectric focusing assay, which creates a pH gradient andmay utilize laser focusing for increased accuracy (Janini et al (2002)Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89;Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it ispreferred to have an anti-PTK7 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range, or by mutating charged surfaceresidues using standard techniques well known in the art.

Each antibody will have a melting temperature that is indicative ofthermal stability (Krishnamurthy R and Manning M C (2002) Curr PharmBiotechnol 3:361-71). A higher thermal stability indicates greateroverall antibody stability in vivo. The melting point of an antibody maybe measure using techniques such as differential scanning calorimetry(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLett 68:47-52). T_(M1) indicates the temperature of the initialunfolding of the antibody. T_(M2) indicates the temperature of completeunfolding of the antibody. Generally, it is preferred that the T_(M1) ofan antibody of the present invention is greater than 60° C., preferablygreater than 65° C., even more preferably greater than 70° C.Alternatively, the thermal stability of an antibody may be measure usingcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not rapidlydegrade. Fragmentation of an anti-PTK7 antibody may be measured usingcapillary electrophoresis (CE) and MALDI-MS, as is well understood inthe art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects. Aggregation may lead to triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation may be measured by several techniques well known in the art,including size-exclusion column (SEC) high performance liquidchromatography (HPLC), and light scattering to identify monomers,dimers, trimers or multimers.

Methods of Engineering Antibodies

As discussed above, the anti-PTK7 antibodies having V_(H) and V_(K)sequences disclosed herein can be used to create new anti-PTK7antibodies by modifying the VH and/or V_(K) sequences, or the constantregion(s) attached thereto. Thus, in another aspect of the invention,the structural features of an anti-PTK7 antibody of the invention, e.g.3G8, 3G8a, 4D5, 12C6, 12C6a or 7C8, are used to create structurallyrelated anti-PTK7 antibodies that retain at least one functionalproperty of the antibodies of the invention, such as binding to humanPTK7. For example, one or more CDR regions of 3G8, 3G8a, 4D5, 12C6,12C6a or 7C8, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-PTK7 antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(K) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(K) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-PTK7 antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence selected from the group        consisting of SEQ ID NOs: 11, 12, 13 and 14, a CDR2 sequence        selected from the group consisting of SEQ ID NOs: 15, 16, 17 and        18, and/or a CDR3 sequence selected from the group consisting of        SEQ ID NOs: 19, 20, 21 and 22; and/or (ii) a light chain        variable region antibody sequence comprising a CDR1 sequence        selected from the group consisting of SEQ ID NOs: 23, 24, 25,        26, 27 and 28, a CDR2 sequence selected from the group        consisting of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, and/or a        CDR3 sequence selected from the group consisting of SEQ ID NOs:        35, 36, 37, 38, 39 and 40;    -   (b) altering at least one amino acid residue within the heavy        chain variable region antibody sequence and/or the light chain        variable region antibody sequence to create at least one altered        antibody sequence; and    -   (c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-PTK7 antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (a) the antibody binds to human PTK7 with a K_(D) of 1×10⁻⁷ M or        less;    -   (b) the antibody binds the Wilms' tumor cell line.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., flow cytometry, bindingassays).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-PTK7 antibody coding sequence and the resultingmodified anti-PTK7 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules of the invention are those encodingthe VH and VL sequences of the 3G8, 3G8a, 4D5, 12C6, 12C6a or 7C8monoclonal antibodies. DNA sequences encoding the VH sequences of 3G8,3G8a, 4D5, 12C6, 12C6a and 7C8 are shown in SEQ ID NOs: 41 (3G8 and3G8a), 42 (4D5), 43 (12C6 and 12C6a) and 44 (7C8). DNA sequencesencoding the VL sequences of 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 areshown in SEQ ID NOs: 45, 46, 47, 48, 49 and 50, respectively.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstPTK7 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM Mice™, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368 (6474): 856-859). Accordingly, the mice exhibitreduced expression of mouse IgM or κ, and in response to immunization,the introduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM Mice™”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PTK7 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PTK7 antibodies of the invention. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-PTK7antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or enriched preparation ofPTK7 antigen and/or recombinant PTK7, or a PTK7 fusion protein, asdescribed by Lonberg, N. et al. (1994) Nature 368 (6474): 856-859;Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; and PCTPublication WO 98/24884 and WO 01/14424. Preferably, the mice will be6-16 weeks of age upon the first infusion. For example, a purified orrecombinant preparation (5-50 μg) of PTK7 antigen can be used toimmunize the human Ig mice intraperitoneally.

Detailed procedures to generate fully human monoclonal antibodies toPTK7 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-PTK7 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12). Alternatively or additionally, the KMMouse™ strain can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3×63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×10⁵in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(K) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to PTK7 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified PTK7 at 0.25 μg/ml in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom PTK7-immunized mice) are added to each well and incubated for 1-2hours at 37° C. The plates are washed with PBS/Tween and then incubatedwith secondary reagent (e.g., for human antibodies, a goat-anti-humanIgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatasefor 1 hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micewhich develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with PTK7 immunogen. Hybridomasthat bind with high avidity to PTK7 are subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-PTK7 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-PTK7 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using PTK7 coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strepavidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-PTK7 human IgGs can be further tested for reactivity with PTK7antigen by Western blotting. Briefly, PTK7 can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugates

In another aspect, the present invention features an anti-PTK7 antibody,or a fragment thereof, conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Suchconjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst). Examples of therapeutic cytotoxins may befound, for example, in U.S. Pat. Nos. 6,548,530 and 6,281,354 and USPatent application Nos: US 2003/0064984, US 2003/0073852 and US2003/0050331.

Cytotoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-PTK7 antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for PTK7 and a secondbinding specificity for a second target epitope. In a particularembodiment of the invention, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing PTK7. These bispecific molecules target PTK7 expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an PTK7 expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

In an embodiment of the invention in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-PTK7 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the F_(c) receptor or target cellantigen. The “anti-enhancement factor portion” can bind an F_(c)receptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγRI(CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFcγ binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol.155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of the invention because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-PTK7 binding specificities, using methods known in the art. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-5-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-PTK7 antibody of the presentinvention combined with at least one other anti-inflammatory orimmunosuppressant agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-PTK7antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μ/ml and in some methods about 25-300μ/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-PTK7 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies, antibody compositions and methods of the presentinvention have numerous in vitro and in vivo diagnostic and therapeuticutilities involving the diagnosis and treatment of PTK7 mediateddisorders. In a preferred embodiment, the antibodies of the presentinvention are human antibodies. For example, these molecules can beadministered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to treat, prevent and to diagnose a variety ofdisorders. As used herein, the term “subject” is intended to includehuman and non-human animals. Non-human animals includes all vertebrates,e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, and reptiles. Preferredsubjects include human patients having disorders mediated by PTK7activity. The methods are particularly suitable for treating humanpatients having a disorder associated with aberrant PTK7 expression.When antibodies to PTK7 are administered together with another agent,the two can be administered in either order or simultaneously.

Given the specific binding of the antibodies of the invention for PTK7,the antibodies of the invention can be used to specifically detect PTK7expression on the surface of cells and, moreover, can be used to purifyPTK7 via immunoaffinity purification.

The invention further provides methods for detecting the presence ofhuman PTK7 antigen in a sample, or measuring the amount of human PTK7antigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to human PTK7, under conditions that allow forformation of a complex between the antibody or portion thereof and humanPTK7. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human PTK7 antigen in the sample.

PTK7 is expressed in colon carcinoma derived cell lines but not found tobe expressed in human adult colon tissues (Mossie et al. (1995) Oncogene11:2179-84). PTK7 expression was also seen in some melanoma cell linesand melanoma biopsies (Easty, et al. (1997) Int. J. Cancer 71:1061-5).In addition, PTK7 was found to be highly overexpressed in acute myeloidleukemia samples (Muller-Tidow et al., (2004) Clin. Cancer Res.10:1241-9). An anti-PTK7 antibody may be used alone to inhibit thegrowth of cancerous tumors. Alternatively, an anti-PTK7 antibody may beused in conjunction with other immunogenic agents, standard cancertreatments or other antibodies, as described below.

Preferred cancers whose growth may be inhibited using the antibodies ofthe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment include coloncancer (including small intestine cancer), lung cancer, breast cancer,pancreatic cancer, melanoma (e.g., metastatic malignant melanoma), acutemyeloid leukemia, kidney cancer, bladder cancer, ovarian cancer andprostate cancer. Examples of other cancers that may be treated using themethods of the invention include renal cancer (e.g., renal cellcarcinoma), glioblastoma, brain tumors, chronic or acute leukemiasincluding acute lymphocytic leukemia (ALL), adult T-cell leukemia(T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin'slymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma,Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneousT-cell lymphomas, nodular small cleaved-cell lymphomas, peripheralT-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cellleukemia/lymphomas (ATLL), entroblastic/centrocytic (cb/cc) follicularlymphomas cancers, diffuse large cell lymphomas of B lineage,angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIVassociated body cavity based lymphomas), embryonal carcinomas,undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke'stumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma,Waldenstrom's macroglobulinemia and other B-cell lymphomas,nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular malignant melanoma, uterine cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, solid tumors ofchildhood, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, epidermoid cancer, squamous cell cancer,environmentally induced cancers including those induced by asbestos,e.g., mesothelioma and combinations of said cancers.

Furthermore, given the expression of PTK7 on various tumor cells, thehuman antibodies, antibody compositions and methods of the presentinvention can be used to treat a subject with a tumorigenic disorder,e.g., a disorder characterized by the presence of tumor cells expressingPTK7 including, for example, colon cancer (including small intestinecancer), melanoma (e.g., metastatic malignant melanoma), acute myeloidleukemia, lung cancer, breast cancer, bladder cancer, pancreatic cancer,ovarian cancer and prostate cancer. Examples of other subjects with atumorigenic disorder include subjects having renal cancer (e.g., renalcell carcinoma), glioblastoma, brain tumors, chronic or acute leukemiasincluding acute lymphocytic leukemia (ALL), adult T-cell leukemia(T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin'slymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma,Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneousT-cell lymphomas, nodular small cleaved-cell lymphomas, peripheralT-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cellleukemia/lymphomas (ATLL), entroblastic/centrocytic (cb/cc) follicularlymphomas cancers, diffuse large cell lymphomas of B lineage,angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIVassociated body cavity based lymphomas), embryonal carcinomas,undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke'stumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma,Waldenstrom's macroglobulinemia and other B-cell lymphomas,nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular malignant melanoma, uterine cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, solid tumors ofchildhood, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, epidermoid cancer, squamous cell cancer,environmentally induced cancers including those induced by asbestos,e.g., mesothelioma and combinations of said cancers.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-PTK7antibody or antigen-binding portion thereof. Preferably, the antibody isa human anti-PTK7 antibody (such as any of the human anti-human PTK7antibodies described herein). Additionally or alternatively, theantibody may be a chimeric or humanized anti-PTK7 antibody.

In one embodiment, the antibodies (e.g., human monoclonal antibodies,multispecific and bispecific molecules and compositions) of theinvention can be used to detect levels of PTK7 or levels of cells whichcontain PTK7 on their membrane surface, which levels can then be linkedto certain disease symptoms. Alternatively, the antibodies can be usedto inhibit or block PTK7 function which, in turn, can be linked to theprevention or amelioration of certain disease symptoms, therebyimplicating PTK7 as a mediator of the disease. This can be achieved bycontacting an experimental sample and a control sample with theanti-PTK7 antibody under conditions that allow for the formation of acomplex between the antibody and PTK7. Any complexes formed between theantibody and PTK7 are detected and compared in the experimental sampleand the control.

In another embodiment, the antibodies (e.g., human antibodies,multispecific and bispecific molecules and compositions) of theinvention can be initially tested for binding activity associated withtherapeutic or diagnostic use in vitro. For example, compositions of theinvention can be tested using the flow cytometric assays described inthe Examples below.

The antibodies (e.g., human antibodies, multispecific and bispecificmolecules, immunoconjugates and compositions) of the invention haveadditional utility in therapy and diagnosis of PTK7-related diseases.For example, the human monoclonal antibodies, the multispecific orbispecific molecules and the immunoconjugates can be used to elicit invivo or in vitro one or more of the following biological activities: toinhibit the growth of and/or kill a cell expressing PTK7; to mediatephagocytosis or ADCC of a cell expressing PTK7 in the presence of humaneffector cells; or to block PTK7 ligand binding to PTK7.

In a particular embodiment, the antibodies (e.g., human antibodies,multispecific and bispecific molecules and compositions) are used invivo to treat, prevent or diagnose a variety of PTK7-related diseases.Examples of PTK7-related diseases include, among others, colon cancer(including small intestine cancer), melanoma (e.g., metastatic malignantmelanoma), acute myeloid leukemia, lung cancer, breast cancer, bladdercancer, pancreatic cancer, ovarian cancer and prostate cancer.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of the invention in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-PTK7 antibodies of the invention canbe co-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/dose once every fourweeks and adriamycin is intravenously administered as a 60-75 mg/ml doseonce every 21 days. Co-administration of the human anti-PTK7 antibodiesor antigen binding fragments thereof, of the present invention withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody.

In one embodiment, immunoconjugates of the invention can be used totarget compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxoins immunosuppressants, etc.) to cells which have PTK7 cellsurface receptors by linking such compounds to the antibody. Forexample, an anti-PTK7 antibody can be conjugated to any of the toxincompounds described in U.S. Pat. Nos. 6,281,354 and 6,548,530, US patentpublication Nos. 20030050331, 20030064984, 20030073852 and 20040087497or published in WO 03/022806, which are hereby incorporated by referencein their entireties. Thus, the invention also provides methods forlocalizing ex vivo or in vivo cells expressing PTK7 (e.g., with adetectable label, such as a radioisotope, a fluorescent compound, anenzyme or an enzyme co-factor). Alternatively, the immunoconjugates canbe used to kill cells which have PTK7 cell surface receptors bytargeting cytotoxins or radiotoxins to PTK7.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used as therapeutic agents.Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10⁸-10⁹ but will vary depending onthe therapeutic purpose. In general, the amount will be sufficient toobtain localization at the target cell, e.g., a tumor cell expressingPTK7 and to effect cell killing by, e.g., phagocytosis. Routes ofadministration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) of the inventionand/or effector cells armed with these compositions can be used inconjunction with chemotherapy. Additionally, combination immunotherapymay be used to direct two distinct cytotoxic effector populations towardtumor cell rejection. For example, anti-PTK7 antibodies linked toanti-Fc-gamma RI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be usedto modulate FcγR or FcγR levels on effector cells, such as by cappingand elimination of receptors on the cell surface. Mixtures of anti-Fcreceptors can also be used for this purpose.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules and immunoconjugates) of the invention which have complementbinding sites, such as portions from IgG1, -2 or -3 or IgM which bindcomplement, can also be used in the presence of complement. In oneembodiment, ex vivo treatment of a population of cells comprising targetcells with a binding agent of the invention and appropriate effectorcells can be supplemented by the addition of complement or serumcontaining complement. Phagocytosis of target cells coated with abinding agent of the invention can be improved by binding of complementproteins. In another embodiment target cells coated with thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be lysed by complement. In yetanother embodiment, the compositions of the invention do not activatecomplement.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules and immunoconjugates) of the invention can also beadministered together with complement. Accordingly, within the scope ofthe invention are compositions comprising human antibodies,multispecific or bispecific molecules and serum or complement. Thesecompositions are advantageous in that the complement is located in closeproximity to the human antibodies, multispecific or bispecificmolecules. Alternatively, the human antibodies, multispecific orbispecific molecules of the invention and the complement or serum can beadministered separately.

Accordingly, patients treated with antibody compositions of theinvention can be additionally administered (prior to, simultaneouslywith or following administration of a human antibody of the invention)with another therapeutic agent, such as a cytotoxic or radiotoxic agent,which enhances or augments the therapeutic effect of the humanantibodies.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fcγ or Fcγ receptors by, for example, treating the subjectwith a cytokine. Preferred cytokines for administration during treatmentwith the multispecific molecule include of granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumornecrosis factor (TNF).

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used to target cells expressingFcγR or PTK7, for example for labeling such cells. For such use, thebinding agent can be linked to a molecule that can be detected. Thus,the invention provides methods for localizing ex vivo or in vitro cellsexpressing Fc receptors, such as FcγR or PTK7. The detectable label canbe, e.g., a radioisotope, a fluorescent compound, an enzyme or an enzymeco-factor.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain one more additionalreagents, such as an immunosuppressive reagent, a cytotoxic agent or aradiotoxic agent or one or more additional human antibodies of theinvention (e.g., a human antibody having a complementary activity whichbinds to an epitope in the PTK7 antigen distinct from the first humanantibody). Kits typically include a label indicating the intended use ofthe contents of the kit. The term label includes any writing, orrecorded material supplied on or with the kit, or which otherwiseaccompanies the kit.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies AgainstPTK7 Antigen

Immunization protocols utilized as antigen both (i) a recombinant fusionprotein comprising the extracellular portion of PTK7 with both a myc andhis tag and (ii) membrane bound full-length PTK7. Both antigens weregenerated by recombinant transfection methods in a CHO cell line.

Transgenic HuMab and KM Mice™

Fully human monoclonal antibodies to PTK7 were prepared using the HCo7and HCo12 strains of HuMab transgenic mice and the KM strain oftransgenic transchromosomic mice, each of which express human antibodygenes. In each of these mouse strains, the endogenous mouse kappa lightchain gene has been homozygously disrupted as described in Chen et al.(1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene hasbeen homozygously disrupted as described in Example 1 of PCT PublicationWO 01/09187. Each of these mouse strains carries a human kappa lightchain transgene, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851. The HCo7 strain carries the HCo7 human heavychain transgene as described in U.S. Pat. Nos. 5,770,429; 5,545,806;5,625,825; and 5,545,807. The HCo12 strain carries the HCo12 human heavychain transgene as described in Example 2 of WO 01/09187 or example 2 WO01/14424. The KM strain contains the SC20 transchromosome as describedin PCT Publication WO 02/43478.

HuMab and KM Immunizations:

To generate fully human monoclonal antibodies to PTK7, HuMab mice and KMMice™ were immunized with purified recombinant PTK7 fusion protein andPTK7-transfected CHO cells as antigen. General immunization schemes forHuMab mice are described in Lonberg, N. et al (1994) Nature 368(6474):856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 andPCT Publication WO 98/24884. The mice were 6-16 weeks of age upon thefirst infusion of antigen. A purified recombinant preparation (5-50 μg)of PTK7 fusion protein antigen and 5-10×10⁶ cells were used to immunizethe HuMab mice and KM Mice™ intraperitonealy, subcutaneously (Sc) or viafootpad injection.

Transgenic mice were immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of11 immunizations) with the antigen in incomplete Freund's or Ribiadjuvant. The immune response was monitored by retroorbital bleeds. Theplasma was screened by ELISA (as described below), and mice withsufficient titers of anti-PTK7 human immunogolobulin were used forfusions. Mice were boosted intravenously with antigen 3 days beforesacrifice and removal of the spleen. Typically, 10-35 fusions for eachantigen were performed. Several dozen mice were immunized for eachantigen.

Selection of HuMab or KM Mice™ Producing Anti-PTK7 Antibodies:

To select HuMab or KM Mice™ producing antibodies that bound PTK7, serafrom immunized mice were tested by ELISA as described by Fishwild, D. etal. (1996). Briefly, microtiter plates were coated with purifiedrecombinant PTK7 fusion protein from transfected CHO cells at 1-2 μg/mlin PBS, 100 μl/wells incubated 4° C. overnight then blocked with 200μl/well of 5% fetal bovine serum in PBS/Tween (0.05%). Dilutions of serafrom PTK7-immunized mice were added to each well and incubated for 1-2hours at ambient temperature. The plates were washed with PBS/Tween andthen incubated with a goat-anti-human IgG polyclonal antibody conjugatedwith horseradish peroxidase (HRP) for 1 hour at room temperature. Afterwashing, the plates were developed with ABTS substrate (Sigma, A-1888,0.22 mg/ml) and analyzed by spectrophotometer at OD 415-495. Mice thatdeveloped the highest titers of anti-PTK7 antibodies were used forfusions. Fusions were performed as described below and hybridomasupernatants were tested for anti-PTK7 activity by ELISA.

Generation of Hybridomas Producing Human Monoclonal Antibodies to PTK7:

The mouse splenocytes, isolated from the HuMab mice, were fused with PEGto a mouse myeloma cell line based upon standard protocols. Theresulting hybridomas were then screened for the production ofantigen-specific antibodies. Single cell suspensions of splenocytes fromimmunized mice were fused to one-fourth the number of SP2/0 nonsecretingmouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells wereplated at approximately 1×10⁵/well in flat bottom microtiter plate,followed by about two week incubation in selective medium containing 10%fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium,3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose,L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, CRL P-7185).After 1-2 weeks, cells were cultured in medium in which the HAT wasreplaced with HT. Individual wells were then screened by ELISA(described above) for human anti-PTK7 monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. The antibody-secreting hybridomas were replated, screenedagain and, if still positive for human IgG, anti-PTK7 monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Hybridoma clones 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 were selected forfurther analysis.

Example 2 Structural Characterization of Human Monoclonal Antibodies3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8

The cDNA sequences encoding the heavy and light chain variable regionsof the 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 monoclonal antibodies wereobtained from the 3G8, 3G8a, 4D5, 12C6, 12C6a and 7C8 hybridomas,respectively, using standard PCR techniques and were sequenced usingstandard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 3G8 are shown in FIG. 1A and in SEQ ID NO:41 and 1,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 3G8 are shown in FIG. 1B and in SEQ ID NO:45 and 5,respectively.

Comparison of the 3G8 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 3G8 heavy chain utilizes a VH segment from human germline VH 3-30.3,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 3G8 VH sequence to the germline VH 3-30.3 sequenceis shown in FIG. 5. Further analysis of the 3G8 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 1A and 5, andin SEQ ID NOs: 11, 15 and 19, respectively.

Comparison of the 3G8 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 3G8 light chain utilizes a VL segment from human germline VK L15 anda JK segment from human germline JK 1. The alignment of the 3G8 VLsequence to the germline VK L15 sequence is shown in FIG. 9. Furtheranalysis of the 3G8 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 1B and 9, and in SEQ ID NOs: 23, 29 and35, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 3G8a are shown in FIG. 1A and in SEQ ID NO:41 and 1,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 3G8a are shown in FIG. 1C and in SEQ ID NO:46 and 6,respectively.

Comparison of the 3G8a heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 3G8a heavy chain utilizes a VH segment from human germline VH3-30.3, an undetermined D segment, and a JH segment from human germlineJH 4b. The alignment of the 3G8a VH sequence to the germline VH 3-30.3sequence is shown in FIG. 5. Further analysis of the 3G8a VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 1A and 5, and in SEQ ID NOs: 11, 15 and 19, respectively.

Comparison of the 3G8a light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 3G8a light chain utilizes a VL segment from human germline VK L15and a JK segment from human germline JK 3. The alignment of the 3G8a VLsequence to the germline VK L15 sequence is shown in FIG. 9. Furtheranalysis of the 3G8a VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 1C and 9, and in SEQ ID NOs: 24, 30 and36, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 4D5 are shown in FIG. 2A and in SEQ ID NO:42 and 2,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 4D5 are shown in FIG. 2B and in SEQ ID NO:47 and 7,respectively.

Comparison of the 4D5 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 4D5 heavy chain utilizes a VH segment from human germline VH 3-30.3,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 4D5 VH sequence to the germline VH 3-30.3 sequenceis shown in FIG. 6. Further analysis of the 4D5 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 2A and 6, andin SEQ ID NOs: 12, 16 and 20, respectively.

Comparison of the 4D5 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 4D5 light chain utilizes a VL segment from human germline VK A10 anda JK segment from human germline JK 5. The alignment of the 4D5 VLsequence to the germline VK A10 sequence is shown in FIG. 10. Furtheranalysis of the 4D5 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 2B and 10, and in SEQ ID NOs: 25, 31 and37, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12C6 are shown in FIG. 3A and in SEQ ID NO:43 and 3,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 12C6 are shown in FIG. 3B and in SEQ ID NO:48 and 8,respectively.

Comparison of the 12C6 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12C6 heavy chain utilizes a VH segment from human germline VH DP44,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 12C6 VH sequence to the germline VH DP44 sequenceis shown in FIG. 7. Further analysis of the 12C6 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 3A and 7, andin SEQ ID NOs: 13, 17 and 21, respectively.

Comparison of the 12C6 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12C6 light chain utilizes a VL segment from human germline VK A27and a JK segment from human germline JK 2. The alignment of the 12C6 VLsequence to the germline VK A27 sequence is shown in FIG. 11. Furtheranalysis of the 12C6 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 11, and in SEQ ID NOs: 26, 32 and38, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12C6a are shown in FIG. 3A and in SEQ ID NO:43 and 3,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 12C6a are shown in FIG. 3C and in SEQ ID NO:49 and 9,respectively.

Comparison of the 12C6a heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12C6a heavy chain utilizes a VH segment from human germline VH DP44,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 12C6a VH sequence to the germline VH DP44 sequenceis shown in FIG. 7. Further analysis of the 12C6a VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 3A and 7, andin SEQ ID NOs: 13, 17 and 21, respectively.

Comparison of the 12C6a light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12C6a light chain utilizes a VL segment from human germline VK L15and a JK segment from human germline JK 2. The alignment of the 12C6a VLsequence to the germline VK L15 sequence is shown in FIG. 12. Furtheranalysis of the 12C6a VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3C and 12, and in SEQ ID NOs: 27, 33 and39, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 7C8 are shown in FIG. 4A and in SEQ ID NO:44 and 4,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 7C8 are shown in FIG. 4B and in SEQ ID NO:50 and 10,respectively.

Comparison of the 7C8 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 7C8 heavy chain utilizes a VH segment from human germline VH 3-33, aD segment from human germline 3-10, and a JH segment from human germlineJH 6b. The alignment of the 7C8 VH sequence to the germline VH 3-33sequence is shown in FIG. 8. Further analysis of the 7C8 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 4A and 8, and in SEQ ID NOs: 14, 18 and 22, respectively.

Comparison of the 7C8 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 7C8 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 3. The alignment of the 7C8 VLsequence to the germline VK L6 sequence is shown in FIG. 13. Furtheranalysis of the 7C8 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 4B and 13, and in SEQ ID NOs: 28, 34 and40, respectively.

Example 3 Mutation of mAb 12C6 and Alternative Germline Usage

As discussed in Example 2 above, monoclonal antibodies 12C6 and 12C6autilize a heavy chain variable region derived from a human DP-44germline sequence present in the HCo7 transgene of the HuMab Mouse®strain. Since DP-44 is not a germline sequence that is utilized in thenative human immunoglobulin repertoire, it may be advantageous to mutatethe VH sequence of 12C6 and 12C6a to reduce potential immunogenicity.Preferably, one or more framework residues of the 12C6 or 12C6a VHsequence is mutated to a residue(s) present in the framework of astructurally related VH germline sequence that is utilized in the nativehuman immunoglobulin repertoire. For example, FIG. 7 shows the alignmentof the 12C6 and 12C6a VH sequence with the DP44 germline sequence andalso to two structurally related human germline sequences, VH 3-23 andVH 3-7. Given the relatedness of these sequences, one can predict thatone can select a human antibody that specifically binds to human PTK7and that utilizes a VH region derived from a VH 3-23 or VH 3-7 germlinesequence. Moreover, one can mutate one or more residues within the 12C6or 12C6a VH sequence that differ from the residue(s) at the comparableposition in the VH 3-23 or VH 3-7 sequence to the residue(s) that ispresent in VH 3-23 or VH 3-7, or to a conservative amino acidsubstitution thereof.

Example 4 Characterization of Binding Specificity and Binding Kineticsof Anti-PTK7 Human Monoclonal Antibodies

In this example, binding affinity and binding kinetics of anti-PTK7antibodies were examined by Biacore analysis. Binding specificity, andcross-competition were examined by flow cytometry.

Binding Specificity by Flow Cytometry

HEK3 cell lines that express recombinant human PTK7 at the cell surfacewere developed and used to determine the specificity of PTK7 humanmonoclonal antibodies by flow cytometry. HEK3 cells were transfectedwith expression plasmids containing full length cDNA encodingtransmembrane forms of PTK7. Binding of the 7C8 anti-PTK7 humanmonoclonal antibody was assessed by incubating the transfected cellswith the anti-PTK7 human monoclonal antibody at a concentration of 10μg/ml. The cells were washed and binding was detected with aFITC-labeled anti-human IgG Ab. Flow cytometric analyses were performedusing a FACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Theresults are depicted in FIG. 14. The anti-PTK7 human monoclonal antibody7C8 bound to the HEK3 cells transfected with PTK7 but not to HEK3 cellsthat were not transfected with human PTK7. This data demonstrates thespecificity of anti-PTK7 human monoclonal antibodies for PTK7.

Binding Specificity by ELISA

The binding of anti-PTK7 antibodies was also assessed by standard ELISAto examine the specificity of binding for PTK7.

Recombinant extracellular domain of PTK7 was tested for binding againstthe anti-PTK7 human monoclonal antibodies 3G8, 4D5, 12C6 and 12C6a atdifferent concentrations. Standard ELISA procedures were performed. Theanti-PTK7 human monoclonal antibodies were added at a startingconcentration of 10 μg/ml and serially diluted at a 1:2 dilution.Goat-anti-human IgG (kappa chain-specific) polyclonal antibodyconjugated with horseradish peroxidase (HRP) was used as secondaryantibody. The results are shown in FIG. 15. Each of the anti-PTK7 humanmonoclonal antibodies 3G8, 4D5, 12C6 and 12C6a bound to PTK7. This datademonstrates the specificity of anti-PTK7 human monoclonal antibodiesfor PTK7.

Epitope Mapping of Anti-PTK7 Antibodies

Flow cytometry was used to determine epitope grouping of anti-PTK7HuMAbs. Wilms' tumor cells G-401 (ATCC Acc No. CRL-1441) weretransfected with expression plasmids containing full length cDNAencoding transmembrane forms of PTK7. Epitope binding of each anti-PTK7human monoclonal antibody was assessed by incubating 1×10⁵ transfectedcells with 10 μg/ml of cold anti-PTK7 human monoclonal antibody, washed,and followed by the addition of 10 μg/ml of a fluorescence-conjugatedanti-PTK7 human monoclonal antibody. Binding was detected with aFITC-labeled anti-human IgG Ab. Flow cytometric analyses were performedusing a FACScan flow cytometry (Becton Dickinson, San Jose, Calif.).Upon analysis of the data, the anti-PTK7 antibodies have beencategorized into 3 epitope groups—group A, which includes 7D11, group B,which includes 3G8 and 3G8a and group C, which includes 7C8, 12C6 and12C6a.

Example 5 Characterization of Anti-PTK7 Antibody Binding to PTK7Expressed on the Surface of Human Cancer Cells

The nephroblastoma Wilms' tumor cell line G-401 (ATCC Acc No. CRL-1441)was tested for binding of the HuMAb anti-PTK7 human monoclonalantibodies 12C6 and 7C8 at different concentrations. Binding of theanti-PTK7 human monoclonal antibodies was assessed by incubating 1×10⁵cells with antibody at a starting concentration of 30 μg/ml and seriallydiluting the antibody at a 1:10 dilution. The cells were washed andbinding was detected with a PE-labeled anti-human IgG Ab. Flowcytometric analyses were performed using a FACScan flow cytometry(Becton Dickinson, San Jose, Calif.). The results are shown in FIG. 16.The anti-PTK7 monoclonal antibodies 12C6 and 7C8 bound to thenephroblastoma Wilms' tumor cell line in a concentration dependentmanner, as measured by the mean fluorescent intensity (MFI) of staining.The EC₅₀ values for the anti-PTK7 monoclonal antibodies 12C6 and 7C8 was4.035 nM and 3.428 nM, respectively.

These data demonstrate that the anti-PTK7 HuMAbs bind to kidney cancercell lines.

Example 6 Binding of Human Anti-PTK7 Antibody to Cancer Cell Lines

Anti-PTK7 antibodies were tested for binding to a variety of cancer celllines by flow cytometry.

Binding of the 3G8, 12C6a, 4D5 and 12C6 anti-PTK7 human monoclonalantibodies to a panel of cancer cell lines was assessed by incubatingcancer cell lines with anti-PTK7 human monoclonal antibodies at aconcentration of 10 μg/ml. The cancer cell lines that were tested wereA-431 (ATCC Acc No. CRL-1555), Wilms tumor cells G-401 (ATCC Acc No.CRL-1441), Saos-2 (ATCC Acc No. HTB-85), SKOV-3 (ATCC Acc No. HTB-77),PC3 (ATCC Acc No. CRL-1435), DMS 114 (ATCC Acc No. CRL-2066), ACHN (ATCCAcc No. CRL-1611), LNCaP (ATCC Acc No. CRL-1740), DU 145 (ATCC Acc No.HTB-81), LoVo (ATCC Acc No. CCL-229) and MIA PaCa-2 (ATCC Acc No.CRL-1420). An isotype control antibody was used as a negative control.The cells were washed and binding was detected with a FITC-labeledanti-human IgG Ab. Flow cytometric analyses were performed using aFACScan flow cytometry (Becton Dickinson, San Jose, Calif.). The resultsare shown in FIG. 17. The anti-PTK7 monoclonal antibodies 3G8, 12C6a,4D5 and 12C6 bound to the cancer cell lines A-431, Wilms tumor cellsG-401, Saos-2, SKOV-3, PC3, DMS 114, ACHN, LNCaP, DU 145, LoVo and MIAPaCa-2, as measured by the mean fluorescent intensity (MFI) of staining.These data demonstrate that the anti-PTK7 HuMAbs bind to a range ofcancer cells that express cell surface PTK7.

Example 7 Binding of Anti-PTK7 to Human T, B and Dendritic Cells

Anti-PTK7 antibodies were tested for binding to CD4+, CD8+ T-cells,CD19+ B-cells and human blood myeloid dendritic cells expressing PTK7 ontheir cell surface by flow cytometry.

Human T cells were activated by anti-CD3 antibody to induce PTK7expression on T cells prior to binding with a human anti-PTK7 monoclonalantibody. Binding of the 7c8 anti-PTK7 human monoclonal antibody wasassessed by incubating the cells with anti-PTK7 human monoclonalantibodies at a concentration of 10 μg/ml. In some experiments, a knownantibody that binds a T and B-cell specific marker was used as apositive control. The cells were washed and binding was detected with aFITC-labeled anti-human IgG Ab. Flow cytometric analyses were performedusing a FACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Theresults are shown in FIG. 18 (activated human T cells and B-cells) and19 (dendritic cells). The anti-PTK7 monoclonal antibody 7C8 bound toactivated human CD4+ and CD8+ T cells and dendritic cells, but not toB-cells, as measured by the mean fluorescent intensity (MFI) ofstaining. These data demonstrate that the anti-PTK7 HuMAbs bind to humanT-cells and dendritic cells.

Example 8 Internalization of Anti-PTK7 Monoclonal Antibody

Anti-PTK7 HuMAbs were tested for the ability to internalize intoPTK7-expressing cell lines using a Hum-Zap internalization assay. TheHum-Zap assay tests for internalization of a primary human antibodythrough binding of a secondary antibody with affinity for human IgGconjugated to the toxin saporin.

The PTK7-expressing cancer cell lines Wilms tumor G-401 (ATCC Acc No.CRL-1441), A-431 (ATCC Acc No. CRL-1555) and PC3 (ATCC Acc No. CRL-1435)were seeded at 1×10⁴ cells/well in 100 μl wells directly. The anti-PTK7.HuMAb antibodies 3G8, 4D5, 12C6 or 7C8 were added to the wells at astarting concentration of 30 nM and titrated down at 1:3 serialdilutions. An isotype control antibody that is non-specific for PTK7 wasused as a negative control. The Hum-Zap (Advanced Targeting Systems, SanDiego, Calif., IT-22-25) was added at a concentration of 11 nM andplates were allowed to incubate for 72 hours. The plates were thenpulsed with 1.0 μCi of ³H-thymidine for 24 hours, harvested and read ina Top Count Scintillation Counter (Packard Instruments, Meriden, Conn.).The results are shown in FIGS. 20A-D. The anti-PTK7 antibodies 3G8, 4D5,12C6 and 7C8 showed an antibody concentration dependent decrease in³H-thymidine incorporation in the PTK7-expressing Wilms' Tumor cancercell line. The anti-PTK7 antibodies 12C6 and 7C8 showed an antibodyconcentration dependent decrease in ³H-thymidine incorporation in thePTK7-expressing cancer cell lines A-431 and PC3. The EC₅₀ value for theanti-PTK7 antibodies 3G8, 4D5, 12C6 and 7C8 in Wilms' tumor cells was0.6437 nM, 0.2516 nM, 0.2053 nM and 0.1788 nM, respectively. The EC₅₀value for the anti-PTK7 antibodies 12C6 and 7C8 in A-431 cells was0.1657 nM and 0.1826 nM, respectively. The EC₅₀ value for the anti-PTK7antibodies 12C6 and 7C8 in PC3 tumor cells was 0.3175 nM and 0.2648 nM,respectively. This data demonstrates that the anti-PTK7 antibodies 3G8,4D5, 12C6 and 7C8 internalize into cancer cells.

Example 9 Assessment of Cell Killing of a Toxin-Conjugated Anti-PTK7Antibody on Human Cancer Cell Lines

In this example, anti-PTK7 monoclonal antibodies conjugated to a toxinwere tested for the ability to kill PTK7+ human cancer cell lines in acell proliferation assay.

The anti-PTK7 HuMAb antibody 12C6a was conjugated to a toxin via alinker, such as a peptidyl, hydrazone or disulfide linker. Examples oftoxin compounds that may be conjugated to the antibodies of the currentinvention are described in US2008/0279868. The PTK7-expressing Wilms'tumor human kidney cancer cell line G-401 (ATCC Acc No. CRL-1441) wasseeded at 10⁴ cells/well in 100 μl wells for 3 hours. An anti-PTK7antibody-toxin conjugate was added to the wells at a startingconcentration of 100 nM and titrated down at 1:3 serial dilutions.Plates were allowed to incubate for 48 hours. The plates were thenpulsed with 1 of ³H-thymidine for 24 hours before termination of theculture, harvested and read in a Top Count Scintillation Counter(Packard Instruments). FIG. 21 shows the effects of the 12C6a-conjugateon the Wilms' tumor cells. The anti-PTK7 antibody 12C6a showed anantibody-toxin concentration dependent decrease in ³H-thymidineincorporation in PTK7-expressing Wilms' tumor human kidney cancer cellline.

This data demonstrates that anti-PTK7 antibodies conjugated to toxinshow specific cytotoxicity to human kidney cancer cells.

Example 10 Assessment of Cell Killing of a Toxin-Conjugated Anti-PTK7Antibody on Human Tumor Cell Lines

In this example, anti-PTK7 monoclonal antibodies conjugated to a toxinwere tested for the ability to kill PTK7⁺ human tumor cell lines havingeither low, intermediate or high cell surface expression of PTK7 in acell proliferation assay.

The anti-PTK7 HuMAb antibody 12C6a was conjugated to a toxin via alinker, such as a peptidyl, hydrazone or disulfide linker. Examples oftoxin compounds that may be conjugated to the antibodies of the currentinvention are described in US2008/0279868. The PTK7-expressing humantumor cancer cell lines A-431, SKOV3, and LoVo were seeded at 10⁴cells/well in 100 μl wells. The cell lines were previously tested forcell surface expression of PTK7 in a standard FACS assay. The A-431 cellline expressed the highest level of PTK7 cell surface expression and theLoVo cell line expressed the lowest level of PTK7 cell surfaceexpression. An anti-PTK7 antibody-toxin conjugate was added to the wellsat a starting concentration of 20 nM and titrated down at 1:2 serialdilutions. An isotype control antibody was used as a negative control.Plates were allowed to incubate for 3 hours and the unbound (free)antibody-toxin conjugates were washed out. The plates continued toincubate for 96 hrs and the killing activity (FU, fluorescent unit) wasmeasured by cell viability in CellTiter-Glo® Luminescent assay accordingto protocol (Promega, Wis., USA, Technical bulletin No. 288) using aBIO-TEK reader (Bio-Tek Instruments, Inc, VT, USA). The results areshown in FIG. 22. The anti-PTK7-toxin conjugate showed an antibody-toxinconcentration dependent decrease in the proliferation assay inPTK7-expressing A431^(high), SKOV3^(inter), and LoVo^(low).

This data demonstrates that anti-PTK7 antibodies conjugated to toxinshow specific cytotoxicity to various human cancer cells.

Example 11 Immunohistochemistry with 3G8, 12C6a, 2E11

The ability of the anti-PTK7 HuMAbs 3G8, 12C6a, and 2E11 to recognizePTK7 by immunohistochemistry was examined using clinical biopsies fromlung cancer, breast cancer, bladder cancer, pancreatic cancer, coloncancer, ovarian cancer, small intestine cancer & prostate cancer.

For immunohistochemistry, 5 μm frozen sections were used (Ardais Inc,USA). After drying for 30 minutes, sections were fixed with acetone (atroom temperature for 10 minutes) and air-dried for 5 minutes. Slideswere rinsed in PBS and then pre-incubated with 10% normal goat serum inPBS for 20 min and subsequently incubated with 10 μg/ml fitcylatedantibody in PBS with 10% normal goat serum for 30 min at roomtemperature. Next, slides were washed three times with PBS and incubatedfor 30 min with mouse anti-FITC (10 μg/ml DAKO) at room temperature.Slides were washed again with PBS and incubated with Goat anti-mouse HRPconjugate (DAKO) for 30 minutes at room temperature. Slides were washedagain 3× with PBS. Diaminobenzidine (Sigma) was used as substrate,resulting in brown staining. After washing with distilled water, slideswere counter-stained with hematoxyllin for 1 min. Subsequently, slideswere washed for 10 secs in running distilled water and mounted inglycergel (DAKO). Clinical biopsy immunohistochemical staining displayedpositive staining in the lung cancer, breast cancer, bladder cancer,pancreatic cancer, colon cancer, ovarian cancer, small intestine cancer& prostate cancer sections. Normal tissue was always negative for PTK7staining whereas within malignant tissue, both cancer activatedfibroblasts and cancerous epithelial cells were observed to be positivefor PTK7 staining. The identity of the cancer activated fibroblasts wasconfirmed in bladder cancer and breast cancer sections by staining witha Fibroblast Activation Protein antibody (FAP, Alexis Biochemicals, SanDiego, USA). FAP is a known marker of cancer activated fibroblasts(Hofheinz et al. (2003) Oncologie 26:44-48).

Example 12 Invasion Assay

In this example, antibodies directed against PTK7 were tested for theability to affect cell invasion in a CHO cell line transfected withPTK7.

The assay was done using a HTS 96-Multiwell Insert System (Cat#351162,BD Biosciences, CA) according to the protocol. Either a CHO parent cellline, CHO cells transfected with full-length PTK7 or a control HEK293cell line were mixed with either a pool of antiPTK7 HuMabs or an isotypecontrol antibody prior to the addition of the cells into the inserts.The mixture (cells+Ab pool) was added into an insert well in theinvasion plate. Following incubation at 37° C. with 5% CO2 for 24 hours,the cells were labeled with a fluorescent dye and cells that invaded tothe bottom of the membrane were quantitated using a fluorescence platereader. The results are shown in FIG. 23. This data demonstrates thatanti-PTK7 antibodies inhibit the invasion mobility of cells expressingPTK7 on the cell surface.

Example 13 Treatment of In Vivo Pancreatic Cancer Cell Xenograft ModelUsing Naked and Cytotoxin-Conjugated Anti-PTK7 Antibodies

This Example discloses the in vivo treatment of mice implanted with apancreatic cell carcinoma tumor with toxin-conjugated anti-PTK7antibodies to examine the in vivo effect of the antibodies on tumorgrowth.

HPAC (human pancreatic adenocarcinoma, ATCC Accession Number CRL-2119)or other suitable pancreatic cancer cells were expanded in vitro usingstandard laboratory procedures. Male Ncr athymic nude mice (Taconic,Hudson, N.Y.) between 6-8 weeks of age were implanted subcutaneously inthe right flank with 2.5×10⁶ HPAC cells in 0.2 ml of PBS/Matrigel (1:1)per mouse. Mice were weighed and measured for tumors three dimensionallyusing an electronic caliper twice weekly after implantation. Tumorvolumes were calculated as height×width×length/2. Mice with HPAC tumorsaveraging 90 mm³ were randomized into treatment groups. The mice wereadministered a single intravenous dose with PBS vehicle, naked anti-PTK7antibody or toxin-conjugated anti-PTK7 HuMAb on Day 0 at the dosageindicated (μmol/kg). Examples of toxin compounds that may be conjugatedto the antibodies of the current invention were described in the pendingU.S. patent application Ser. No. 11/134,826 and the pending U.S. PatentApplication designated MEDX-0034US4. Mice were monitored for tumorgrowth for 61 days post dosing. Mice were euthanized when the tumorsreached tumor end point (2000 mm³) or ulcerated. Anti-PTK7 antibodiesconjugated to a toxin slowed tumor growth progression. The results areshown in FIG. 24. The anti-tumor effect of the anti-PTK7 toxin conjugatewas dose dependent, with the greatest effect seen at a dose of 0.3mmol/kg. Treatment with anti-PTK7 toxin conjugate was well tolerated,with subjects never experiencing greater than 5% median body weight loss(data not shown). Thus, treatment with an anti-PTK7 antibody-toxinconjugate has a direct in vivo inhibitory effect on pancreatic cancertumor growth.

Example 14 Treatment of In Vivo Breast Cancer Cell Xenograft Model UsingNaked and Cytotoxin-Conjugated Anti-PTK7 Antibodies

This Example discloses the in vivo treatment of mice implanted with anadriamycin resistance breast carcinoma tumor with toxin-conjugatedanti-PTK7 antibodies to examine the in vivo effect of the antibodies ontumor growth.

MCF7-adr (human breast cancer cell line resistant to adriamycin) wereexpanded in vitro using standard laboratory procedures. Female CB17.5CIDmice (Taconic, Hudson, N.Y.) between 6-8 weeks of age were implantedsubcutaneously with 1.7 mg 90-day release estrogen pellets, 3.0 mm size(Innovative Research of America, Sarasota, Fla.) at the neck region oneday prior to being implanted subcutaneously in the right flank with10×10⁶ MCF7-Adr cells in 0.2 ml of PBS/Matrigel (1:1) per mouse. Micewere weighed and measured for tumors three dimensionally using anelectronic caliper twice weekly after implantation. Tumor volumes werecalculated as height×width×length/2. Mice with MCF7-adr tumors averaging160 mm³ were randomized into treatment groups. The mice wereadministered a single intravenous dose at 0.1 μmol/kg with PBS vehicle,naked anti-PTK7 antibody or toxin-conjugated anti-PTK7 HuMAb on Day 0.Examples of toxin compounds that may be conjugated to the antibodies ofthe current invention were described in the pending U.S. patentapplication Ser. No. 11/134,826 and the pending U.S. Patent Applicationdesignated MEDX-0034US4. Mice were monitored for tumor growth for 63days post dosing. Mice were euthanized when the tumors were ulcerated.The results are shown in FIG. 25. Anti-PTK7 antibody toxin conjugatesslowed tumor growth progression. Thus, treatment with an anti-PTK7antibody-toxin conjugate has a direct in vivo inhibitory effect onbreast cancer tumor growth.

SEQ ID NO: SEQUENCE 1 VH a.a. 3G8, 3G8a 2 VH a.a. 4D5 3 VH a.a. 12C6,12C6a 4 VH a.a. 7C8 5 VK a.a. 3G8 6 VK a.a. 3G8a 7 VK a.a. 4D5 8 VK a.a.12C6 9 VK a.a. 12C6a 10 VK a.a. 7C8 11 VH CDR1 a.a. 3G8 12 VH CDR1 a.a.4D5 13 VH CDR1 a.a. 12C6 14 VH CDR1 a.a. 7C8 15 VH CDR2 a.a. 3G8 16 VHCDR2 a.a. 4D5 17 VH CDR2 a.a. 12C6 18 VH CDR2 a.a. 7C8 19 VH CDR3 a.a.3G8 20 VH CDR3 a.a. 4D5 21 VH CDR3 a.a. 12C6 22 VH CDR3 a.a. 7C8 23 VKCDR1 a.a. 3G8 24 VK CDR1 a.a. 3G8a 25 VK CDR1 a.a. 4D5 26 VK CDR1 a.a.12C6 27 VK CDR1 a.a. 12C6a 28 VK CDR1 a.a. 7C8 29 VK CDR2 a.a. 3G8 30 VKCDR2 a.a. 3G8a 31 VK CDR2 a.a. 4D5 32 VK CDR2 a.a. 12C6 33 VK CDR2 a.a.12C6a 34 VK CDR2 a.a. 7C8 35 VK CDR3 a.a. 3G8 36 VK CDR3 a.a. 3G8a 37 VKCDR3 a.a. 4D5 38 VK CDR3 a.a. 12C6 39 VK CDR3 a.a. 12C6a 40 VK CDR3 a.a.7C8 41 VH n.t. 3G8, 3G8a 42 VH n.t. 4D5 43 VH n.t. 12C6, 12C6a 44 VHn.t. 7C8 45 VK n.t. 3G8 46 VK n.t. 3G8a 47 VK n.t. 4D5 48 VK n.t. 12C649 VK n.t. 12C6a 50 VK n.t. 7C8 51 VH 3-30.3 germline a.a. 52 VH DP44germline a.a. 53 VH 3-33 germline a.a. 54 VK L15 germline a.a. 55 VK A10germline a.a. 56 VK A27 germline a.a. 57 VK L6 germline a.a. 58 PTK7a.a. 59 JH4b germline a.a 60 JH4b germline a.a. 61 3-7 germline a.a. 623-23 germline a.a. 63 JH4b germline a.a 64 JH6b germline a.a. 65 JK1germline a.a. 66 JK5 germline a.a. 67 JK2 germline a.a. 68 JK2 germlinea.a. 69 JK3 germline a.a.

1. An isolated nucleic acid molecule encoding an antibody orantigen-binding portion thereof, wherein the antibody: a) specificallybinds to human PTK7; and b) binds to a Wilms' tumor cell line (ATCC AccNo. CRL-1441).
 2. The nucleic acid of claim 1, wherein the antibodycomprises a heavy chain variable region encoded by a nucleic acidsequence selected from the group consisting of: SEQ ID NOs: 41, 42, 43,and
 44. 3. The nucleic acid of claim 1, wherein the antibody comprises alight chain variable region encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, and
 50. 4.The nucleic acid molecule of claim 1, wherein the antibody comprisesheavy and light chain variable regions encoded by nucleic acid sequencesselected from the group consisting of SEQ ID NO: 41 and SEQ ID NO: 45;SEQ ID NO: 41 and SEQ ID NO: 46; SEQ ID NO: 42 and SEQ ID NO: 47; SEQ IDNO: 43 and SEQ ID NO: 48; SEQ ID NO: 43 and SEQ ID NO: 49; and SEQ IDNO: 44 and SEQ ID NO: 50, respectively.
 5. An expression vectorcomprising the nucleic acid molecule of claim
 1. 6. A host cellcomprising the expression vector of claim
 5. 7. A method for preparingan anti-PTK7 antibody which comprises expressing the antibody in thehost cell of claim 6 and isolating the antibody from the host cell.
 8. Amethod of treating or preventing a disease characterized by growth oftumor cells expressing PTK7, comprising administering to a subject anantibody or antigen-binding portion thereof, wherein the antibodyspecifically binds to human PTK7 and comprises heavy and light chainvariable region sequences selected from the group consisting of: a) aheavy chain variable region CDR1 comprising SEQ ID NO:11; a heavy chainvariable region CDR2 comprising SEQ ID NO:15; a heavy chain variableregion CDR3 comprising SEQ ID NO:19; a light chain variable region CDR1comprising SEQ ID NO:23; a light chain variable region CDR2 comprisingSEQ ID NO:29; and a light chain variable region CDR3 comprising SEQ IDNO:35; b) a heavy chain variable region CDR1 comprising SEQ ID NO:11; aheavy chain variable region CDR2 comprising SEQ ID NO:15; a heavy chainvariable region CDR3 comprising SEQ ID NO:19; a light chain variableregion CDR1 comprising SEQ ID NO:24; a light chain variable region CDR2comprising SEQ ID NO:30; and a light chain variable region CDR3comprising SEQ ID NO:36; c) a heavy chain variable region CDR1comprising SEQ ID NO:12; a heavy chain variable region CDR2 comprisingSEQ ID NO:16; a heavy chain variable region CDR3 comprising SEQ IDNO:20; a light chain variable region CDR1 comprising SEQ ID NO:25; alight chain variable region CDR2 comprising SEQ ID NO:31; and a lightchain variable region CDR3 comprising SEQ ID NO:37; d) a heavy chainvariable region CDR1 comprising SEQ ID NO:13; a heavy chain variableregion CDR2 comprising SEQ ID NO:17; a heavy chain variable region CDR3comprising SEQ ID NO:21; a light chain variable region CDR1 comprisingSEQ ID NO:26; a light chain variable region CDR2 comprising SEQ IDNO:32; and a light chain variable region CDR3 comprising SEQ ID NO:38;e) a heavy chain variable region CDR1 comprising SEQ ID NO:13; a heavychain variable region CDR2 comprising SEQ ID NO:17; a heavy chainvariable region CDR3 comprising SEQ ID NO:21; a light chain variableregion CDR1 comprising SEQ ID NO:27; a light chain variable region CDR2comprising SEQ ID NO:33; and a light chain variable region CDR3comprising SEQ ID NO:39; and f) a heavy chain variable region CDR1comprising SEQ ID NO:14; a heavy chain variable region CDR2 comprisingSEQ ID NO:18; a heavy chain variable region CDR3 comprising SEQ IDNO:22; a light chain variable region CDR1 comprising SEQ ID NO:28; alight chain variable region CDR2 comprising SEQ ID NO:34; and a lightchain variable region CDR3 comprising SEQ ID NO:40; in an amounteffective to treat or prevent the disease.
 9. A method of treating orpreventing a disease characterized by growth of tumor cells expressingPTK7, comprising administering to a subject an antibody orantigen-binding portion thereof, wherein the antibody specifically bindsto human PTK7 and comprises heavy and light chain variable regionsequences selected from the group consisting of: a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:1 and/ora light chain variable region comprising the amino acid sequence of SEQID NO:5; b) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:1 and/or a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:6; c) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:2 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO:7; d) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:3 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO:8; e) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:3 and/or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:9; and f) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:4 and/ora light chain variable region comprising the amino acid sequence of SEQID NO:10; in an amount effective to treat or prevent the disease.
 10. Amethod of treating or preventing a disease characterized by growth oftumor cells expressing PTK7, comprising administering to a subject anantibody or antigen-binding portion thereof, wherein the antibodycompetes for binding to human PTK7 with an antibody comprising heavy andlight chain variable region sequences selected from the group consistingof: a) a heavy chain variable region CDR1 comprising SEQ ID NO:11; aheavy chain variable region CDR2 comprising SEQ ID NO:15; a heavy chainvariable region CDR3 comprising SEQ ID NO:19; a light chain variableregion CDR1 comprising SEQ ID NO:23; a light chain variable region CDR2comprising SEQ ID NO:29; and a light chain variable region CDR3comprising SEQ ID NO:35; b) a heavy chain variable region CDR1comprising SEQ ID NO:11; a heavy chain variable region CDR2 comprisingSEQ ID NO:15; a heavy chain variable region CDR3 comprising SEQ IDNO:19; a light chain variable region CDR1 comprising SEQ ID NO:24; alight chain variable region CDR2 comprising SEQ ID NO:30; and a lightchain variable region CDR3 comprising SEQ ID NO:36; c) a heavy chainvariable region CDR1 comprising SEQ ID NO:12; a heavy chain variableregion CDR2 comprising SEQ ID NO:16; a heavy chain variable region CDR3comprising SEQ ID NO:20; a light chain variable region CDR1 comprisingSEQ ID NO:25; a light chain variable region CDR2 comprising SEQ IDNO:31; and a light chain variable region CDR3 comprising SEQ ID NO:37;d) a heavy chain variable region CDR1 comprising SEQ ID NO:13; a heavychain variable region CDR2 comprising SEQ ID NO:17; a heavy chainvariable region CDR3 comprising SEQ ID NO:21; a light chain variableregion CDR1 comprising SEQ ID NO:26; a light chain variable region CDR2comprising SEQ ID NO:32; and a light chain variable region CDR3comprising SEQ ID NO:38; e) a heavy chain variable region CDR1comprising SEQ ID NO:13; a heavy chain variable region CDR2 comprisingSEQ ID NO:17; a heavy chain variable region CDR3 comprising SEQ IDNO:21; a light chain variable region CDR1 comprising SEQ ID NO:27; alight chain variable region CDR2 comprising SEQ ID NO:33; and a lightchain variable region CDR3 comprising SEQ ID NO:39; and f) a heavy chainvariable region CDR1 comprising SEQ ID NO:14; a heavy chain variableregion CDR2 comprising SEQ ID NO:18; a heavy chain variable region CDR3comprising SEQ ID NO:22; a light chain variable region CDR1 comprisingSEQ ID NO:28; a light chain variable region CDR2 comprising SEQ IDNO:34; and a light chain variable region CDR3 comprising SEQ ID NO:40.11. The method of claim 8, wherein the cancer is selected from the groupconsisting of colon cancer, lung cancer, breast cancer, pancreaticcancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer,ovarian cancer and prostate cancer.